Product Name:	Mouse B4galt1 ELISA Kit
Product Code:	MOFI00620
Size:	96 Assays
Alias:	B4galt1, GGTB2, 1, 4- galactosyltransferase, polypeptide 1, B4GAL-T1, b4Gal-T1, beta-1, 4-galactosyltransferase 1, Beta-1, 4-GalTase 1, Beta4Gal-T1, beta4Gal-T1, betaGlcNAc beta, CDG2D, glycoprotein-4-beta-galactosyltransferase 2, GT1, GTB, lactose synthase, UDP-Gal, UDP-Gal:beta-GlcNAc beta-1, 4-galactosyltransferase 1, UDP-galactose:beta-N-acetylglucosamine beta-1, 4-galactosyltransferase 1
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse B4galt1 concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse B4galt1 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse B4galt1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-105	97
EDTA plasma(n=5)	87-102	95
UFH plasma(n=5)	88-101	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse B4galt1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	93-105%	93-101%	85-100%
EDTA plasma(n=5)	82-98%	89-98%	87-98%
UFH plasma(n=5)	81-97%	82-98%	81-93%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8-12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P15535

UniProt Protein Function:	B4GALT1: The Golgi complex form catalyzes the production of lactose in the lactating mammary gland and could also be responsible for the synthesis of complex-type N-linked oligosaccharides in many glycoproteins as well as the carbohydrate moieties of glycolipids. Defects in B4GALT1 are the cause of congenital disorder of glycosylation type 2D (CDG2D). CDGs are a family of severe inherited diseases caused by a defect in protein N- glycosylation. They are characterized by under-glycosylated serum proteins. These multisystem disorders present with a wide variety of clinical features, such as disorders of the nervous system development, psychomotor retardation, dysmorphic features, hypotonia, coagulation disorders, and immunodeficiency. The broad spectrum of features reflects the critical role of N-glycoproteins during embryonic development, differentiation, and maintenance of cell functions. Belongs to the glycosyltransferase 7 family. 2 isoforms of the human protein are produced by alternative initiation.
UniProt Protein Details:	Protein type:EC 2.4.1.90; Glycan Metabolism - N-glycan biosynthesis; Cell adhesion; Membrane protein, integral; Glycan Metabolism - keratan sulfate biosynthesis; EC 2.4.1.22; Motility/polarity/chemotaxis; Cell surface; EC 2.4.1.38; Transferase; Cell development/differentiation; Carbohydrate Metabolism - galactose; Glycan Metabolism - glycosphingolipid biosynthesis - lacto and neolacto series Chromosomal Location of Human Ortholog: 9p13 Cellular Component: glycocalyx; Golgi apparatus; desmosome; extracellular space; Golgi trans cisterna; basolateral plasma membrane; brush border membrane; integral to membrane; Golgi membrane; membrane; plasma membrane; filopodium; external side of plasma membrane Molecular Function:beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase activity; protein homodimerization activity; N-acetyllactosamine synthase activity; manganese ion binding; lactose synthase activity; cytoskeletal protein binding; beta-tubulin binding; UDP-galactosyltransferase activity; galactosyltransferase activity; alpha-tubulin binding Biological Process: keratan sulfate metabolic process; extracellular matrix organization and biogenesis; glycosaminoglycan metabolic process; positive regulation of epithelial cell proliferation involved in wound healing; oligosaccharide biosynthetic process; positive regulation of apoptosis involved in mammary gland involution; regulation of acrosome reaction; pathogenesis; lactose biosynthetic process; negative regulation of cell proliferation; mammary gland development; development of secondary sexual characteristics; single fertilization; penetration of zona pellucida; epithelial cell development; cell adhesion; Notch signaling pathway; binding of sperm to zona pellucida; protein amino acid N-linked glycosylation; regulation of cell motility; angiogenesis involved in wound healing; post-translational protein modification; multicellular organism reproduction; cellular protein metabolic process; branching morphogenesis of a tube; keratan sulfate biosynthetic process; carbohydrate metabolic process; galactose metabolic process; protein amino acid N-linked glycosylation via asparagine; leukocyte migration; acute inflammatory response Disease: Congenital Disorder Of Glycosylation, Type Iid
NCBI Summary:	This gene is one of seven beta-1,4-galactosyltransferase (beta4GalT) genes. They encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta1,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl. Each beta4GalT has a distinct function in the biosynthesis of different glycoconjugates and saccharide structures. As type II membrane proteins, they have an N-terminal hydrophobic signal sequence that directs the protein to the Golgi apparatus and which then remains uncleaved to function as a transmembrane anchor. By sequence similarity, the beta4GalTs form four groups: beta4GalT1 and beta4GalT2, beta4GalT3 and beta4GalT4, beta4GalT5 and beta4GalT6, and beta4GalT7. This gene is unique among the beta4GalT genes because it encodes an enzyme that participates both in glycoconjugate and lactose biosynthesis. For the first activity, the enzyme adds galactose to N-acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains. The second activity is restricted to lactating mammary tissues where the enzyme forms a heterodimer with alpha-lactalbumin to catalyze UDP-galactose + D-glucose <=> UDP + lactose. The two enzymatic forms result from alternate transcription initiation sites and post-translational processing. Two transcripts, which differ only at the 5' end, with approximate lengths of 4.1 kb and 3.9 kb encode the same protein. The longer transcript encodes the type II membrane-bound, trans-Golgi resident protein involved in glycoconjugate biosynthesis. The shorter transcript encodes a protein which is cleaved to form the soluble lactose synthase. [provided by RefSeq, Jul 2008]
UniProt Code:	P15535
NCBI GenInfo Identifier:	13929462
NCBI Gene ID:	2683
NCBI Accession:	NP_001488.2
UniProt Secondary Accession:	P15535,Q12909, Q12910, Q12911, Q14456, Q14509, Q14523 B2R710, D3DRL2,
UniProt Related Accession:	P15291
Molecular Weight:	43920
NCBI Full Name:	beta-1,4-galactosyltransferase 1
NCBI Synonym Full Names:	UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1
NCBI Official Symbol:	B4GALT1
NCBI Official Synonym Symbols:	GT1; GTB; CDG2D; GGTB2; B4GAL-T1; beta4Gal-T1
NCBI Protein Information:	beta-1,4-galactosyltransferase 1
UniProt Protein Name:	Beta-1,4-galactosyltransferase 1
UniProt Synonym Protein Names:	UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 1; UDP-galactose:beta-N-acetylglucosamine beta-1,4-galactosyltransferase 1
Protein Family:	Beta-1,4-galactosyltransferase
UniProt Gene Name:	B4GALT1
UniProt Entry Name:	B4GT1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 - g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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Mouse B4galt1 ELISA Kit

Description

Overview

Additional Information

Kit Components

Protein Information

Protocol

Sample Preparation